The cadherin family of molecules consists of transmembrane glycoproteins that function in calcium dependent, selective cell-cell interactions. These molecules play important roles during embryonic development and tissue morphogenesis by mediating cell recognition and cell sorting. Subfamilies of cadherins (classic cadherins, protocadherins, desmocollins, and other cadherin-related proteins) are characterized by variable numbers of extracellular cadherin domains, a single transmembrane segment, and a single cytoplasmic domain. The so-called classic cadherins (i.e., E, P, N, and R-cadherin) reportedly have five tandemly repeated extracellular cadherin domains (EC1-EC5) that engage in preferentially homophillic interactions, and a highly conserved cytoplasmic tail that mediates adhesion specific intracellular signaling.
Cadherin mediated cell-cell adhesions occur as multiple cadherin molecules expressed on adjacent cells interact, leading to the formation of adherens junctions. According to the cadherin zipper model proposed by Shapiro et al. Nature 1995; 374(6520):327-37, cadherin molecules within the membrane of the same cell form tight parallel-strand dimers (i.e., so-called cis-dimers). As illustrated in FIG. 1, these cis-dimers then bind to cadherin dimers expressed on adjacent cells (i.e., trans-dimerization). Once a sufficient interaction is sustained, cadherin clustering can occur as more cadherin molecules are recruited to the site, leading to interdigitation of molecules from the two-cell surfaces. In this manner, relatively weak interactions can combine to form fairly strong cell-cell adhesions.
Upon initial cadherin adhesion, intracellular signals, transmitted through interactions of the cytoplasmic cadherin tails with α and β catenin molecules, lead to reorganization of the cytoskeleton. Although the association with actin filaments is not thought to affect homophillic binding, their association helps hold the cadherin molecules at the sites of interaction. In a symbiotic-type of relationship, cadherin clustering causes reorganization of the cytoskeleton and provides points of attachment at the membrane, which are important for cellular changes that occur upon the formation of adherens junctions. Meanwhile, association with the cytoskeleton holds cadherins at the sites of interaction and helps recruit new cadherin molecules, thus mediating cadherin clustering. Calcium plays an important role as a cofactor during cadherin clustering. Cadherin function is lost and molecules become more susceptible to protease degradation in solutions with insufficient concentrations of calcium ions (i.e., below about 2 mM). This is due to the requirement of calcium to stabilize the structure of cadherin molecules and provide proper orientation of adjacent cadherin interfaces.
Although it is reported that each of the five extracellular classical cadherin domains EC1 through EC5 plays an important role in mediating cadherin dimerization, mutational analysis has suggested that the majority of residues that form the dimerization interface are found within the N-terminal most cadherin domain (EC1) (Kitagawa, et al., Biochem. Biophys. Res. Commun., 2000; 271(2):358-63.). However, relatively little is known about the mechanisms of specific homodimerization between cadherin molecules.
Cadherins play a significant role during neuronal guidance and development of the central nervous system. Different subdivisions of the brain are reportedly defined by differential expression of cadherin types. Cadherins also play an important role in neural retinal development by specific expression within different regions of the developing retina. For example, during embryonic chick retina development, B-cadherin is reportedly only found in Müller glia, while certain populations of bipolar cells express R-cadherin (also known as cadherin-4). Amacrine cells and a subset of ganglion cells express cadherins 6B and 7. Within the inner plexiform layer of the retina, these same cadherins are only expressed in sublaminae associated with synapsin-I positive nerve terminals, suggesting that distinct expression profiles contribute to synapse formation between specific subpopulations of neurons during retina development. In the embryonic optic nerve, ganglion cell axon outgrowth is mediated by N-cadherin adhesion with R-cadherin-expressing glial cells.
Cadherin adhesion also plays a role in developmental retinal vascularization (Dorrell, et al. Invest. Ophthalmol. Vis. Sci. 2002; 43(11):3500-10). Disruption of R-cadherin adhesion during formation of the superficial vascular plexus results in the loss of complex vascular interconnections observed during normal vascular patterning. When R-cadherin adhesion is blocked during the subsequent formation of deep vascular layers, key guidance cues are lost causing the vessels to migrate past the normal deep vascular plexuses and into the photoreceptor layer.
The retina consists of well-defined layers of neuronal, glial, and vascular elements. Any disease or condition that alters the retinal layers even slightly, can lead to neuronal degeneration and significant visual loss. The retinal degeneration mouse (rd/rd mouse) has been investigated for over 70 years as a model for many diseases that lead to photoreceptor cell death. In the rd/rd mouse, photoreceptor degeneration begins during the first three weeks after birth as rod cells undergo apoptosis, attributed to a mutation in the β subunit of cGMP-dependent phosphodiesterase followed by cone photoreceptor death. Vascular atrophy within the retina is temporally associated with photoreceptor cell death in rd/rd mice as well. The vasculature appears to form in the normal characteristic fashion as three functional layers develop within the first three weeks. However, the vessels in the deep vascular layer begin to degenerate during the second week and by the end of the fourth postnatal week, dramatic vascular reduction is observed as the deep and intermediate plexuses nearly completely disappear.
A population of hematopoietic stem cells resides in the normal adult circulation and bone marrow, from which different sub-populations of cells can differentiate along lineage positive (Lin+HSC) or lineage negative (Lin−HSC) lineages. In addition, the present inventors have discovered that endothelial precursor cells (EPCs), capable of forming blood vessels in vitro and in vivo, are present within the Lin−HSC subpopulation. EPCs within the population of Lin−HSCs can target and stabilize the degenerating vasculature in rd/rd mice when injected intravitrally to the eyes of the mice. Intravitreally injected Lin−HSCs target astrocytes in the superficial vascular layer and are observed ahead of the endogenous developing vascular network when injected at postnatal day 2 (P2). As the endogenous vasculature reaches the periphery of the retina, where the Lin−HSCs have targeted, the cells are incorporated into new blood vessels, forming functional mosaic vessels with mixed populations of injected Lin−HSCs and endogenous endothelial cells. In addition, Lin−HSCs target the regions of deep and intermediate vascular layers of the retina before vascularization of these layers by endogenous endothelial cells had occurs. Incorporation of Lin−HSCs rescues the deep vasculature of rd/rd mice about 2 to about 3 fold over normal and control Lin+HSC injected mice. In addition, rescue of the deep vasculature prevents degradation of photoreceptors in the outer nuclear layer of the retina. However, as there is no evidence to suggest that these stem cells can undergo differentiation into retinal neurons or glial cells, the mechanism of neuronal protection remains unknown.
The targeting of Lin−HSCs to the astrocytes and deep vascular regions ahead of natural developmental vascularization suggests that the Lin−HSCs express cell-surface molecules that function in targeting, similar to targeting of the endogenous endothelial cells during development. R-cadherin adhesion plays an important role in endothelial cell targeting to astrocytes and vascular plexuses during developmental retinal angiogenesis.
R-cadherin has been identified and sequenced in a number of mammals. FIG. 2 depicts the amino acid sequence (SEQ ID NO: 1) of a human variant of R-cadherin preproprotein reported by Kitagawa et al. in the SWISS-PROT database as Accession No. NP 001785, version NP 001785.2, GI:14589893, the relevant disclosure of which is incorporated herein by reference. SEQ ID NO: 1 includes the amino acid sequence IDSMSGR (SEQ ID NO: 2) at positions 222-228.
FIG. 3 depicts the amino acid sequence (SEQ ID NO: 3) of another human variant of R-cadherin preproprotein reported by Tanihara et al. in the SWISS-PROT database as Accession No. P55283, version P55283, GI:1705542, the relevant disclosure of which is incorporated herein by reference. SEQ ID NO: 3 includes the amino acid sequence INSMSGR (SEQ ID NO: 4) at positions 222-228.
FIG. 4 depicts the amino acid sequence (SEQ ID NO: 5) of a murine (mus musculus) variant of R-cadherin preproprotein reported by Hutton et al. in the SWISS-PROT database as Accession No. NP 033997, version NP 033997.1, GI:6753376, the relevant disclosure of which is incorporated herein by reference. SEQ ID NO: 5 includes the amino acid sequence IDSMSGR (SEQ ID NO: 2) at positions 219-225.
Non-selective peptide antagonists of cadherins including the amino acid sequence His-Ala-Val (HAV) have been reported by Blaschuk et al. in U.S. Pat. No. 6,465,427, U.S. Pat. No. 6,3456,512, U.S. Pat. No. 6,169,071, and U.S. Pat. No. 6,031,072. Blaschuk et al. have reported both linear and cyclic peptide antagonists of cadherins, all of which are capable of antagonizing a number of types of cadherin molecules indiscriminately.
Selective peptide antagonists of N-cadherin, which comprise the amino acid sequence Ile-Asn-Pro (INP) have been reported by Williams et al., Mol. Cell Neurosci., 2000;15(5):456-64. While HAV peptides are non-specific cadherin antagonists, the INP peptide antagonists reported by Williams et al. are specific for N-cadherin and do not exhibit significant binding to other cadherin molecules such as R-cadherin.
Because of the differential distribution of cell adhesion molecules in various tissues in the body, there is an ongoing need for antagonists that are highly selective for specific cell adhesion molecules, in particular for antagonists that are selective for R-cadherin. The selective R-cadherin antagonist peptides of the present invention fulfill this need.